Developing functionally graded PVA hydrogel using simple freeze-thaw method for artificial glenoid labrum

Journal of the Mechanical Behavior of Biomedical Materials 91 (2019) 406–415

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Developing functionally graded PVA hydrogel using simple freeze-thaw method for artificial glenoid labrum

T

Abdul Hadi Abdul Wahaba, Amir Putra Md Saada, Muhammad Noor Harunb, Ardiyansyah Syahroma, Muhammad Hanif Ramleea, Mohd Ayob Sulongb, ⁎ Mohammed Rafiq Abdul Kadira, a

Medical Devices Technology Centre (Meditec), Faculty of Engineering, School of Biomedical Engineering & Health Sciences, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia b Sport Innovation and Technology Centre (SITC), Institute of Human Centred Engineering (IHCE), Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

ARTICLE INFO

ABSTRACT

Keywords: Artificial glenoid labrum PVA hydrogel Freeze-thaw method Gradual stiffness Compressive modulus

Intact glenoid labrum is one of passive stabilizer for glenohumeral joint, which have various stiffness at different region. The aim of this study is to develop new artificial glenoid labrum from Polyvinyl Alcohol (PVA) hydrogel, which known as good biomaterial due to its biocompatibility and ability to tailor its modulus. PVA hydrogel was formed using freeze-thaw (FT) method and the stiffness of PVA was controlled by manipulating the concentration of PVA and number of FT cycles. Then, the gradual stiffness was formed using simple diffusion method by introducing the pre-freeze-and-thaw steps. The results showed 20% PVA with three FT cycles suit to highest stiffness of glenoid labrum while 10% PVA with three FT cycles suit to lowest stiffness of glenoid labrum. The functionally graded PVA hydrogel was then developed using the same method by diffusing two mixture (20% PVA and 10% PVA). Mechanical compression test showed, the highest modulus (0.41 MPa) found at the 20% PVA region and lowest modulus (0.1 MPa) found at 10% PVA region. While, at intermediate region, the compressive modulus was in between 20% and 10%, 0.2 MPa. The existence of gradual stiffness was further prove by checking crystallinity of material at each region using Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD). Microstructure of material was obtained from Scanning Electron Microscopy (SEM). This functionally graded PVA hydrogel also able to reduce about 51% of stress at glenoid implant and up to 17% for micromotion at the interfaces. Existence of artificial glenoid labrum could minimize the occurrence of glenoid component loosening.

1. Introduction Intact glenoid labrum provides 10% stability to glenohumeral joint (Halder et al., 2001) by deepening the curvatures up to 50% of glenoid bone curvature (Howell and Galinat, 1989; Rockwood et al., 2009). Exclusion of glenoid labrum becomes a root problem that leads to most common complication after shoulder replacement (known as glenoid loosening) that induced high stress at the implant. Consequently, it could lead to two possible problems; wear and Rocking Horse Phenomena (RHP) (Matsen et al., 2008). To date, there is no study had been done to produce artificial glenoid labrum due to different stiffness at different region as reported by Carey et al. (2000). Urgently, an artificial glenoid labrum should be considered in using concept of functionally graded material. Hypothetically, this functionally graded

material could resolve the high stress issues in glenoid implant. The main stabilizer at glenohumeral joint (ball-and-socket joint, and look alike a golf ball on a tee) provided by combination of four muscles namely, supraspinatus, infraspinatus, teres minor, and subscapularis. Furthermore, additional passive stabilizer are provide by glenoid labrum (a fibrocartilage surround the glenoid bone) which deepen the curvature up to 50% of the glenoid bone curvature (Howell and Galinat, 1989; Rockwood et al., 2009). It helps to stabilize up to 10% (Halder et al., 2001), glenohumeral joint by acting as an absorber and prevent from excessive humeral head translation (Howell and Galinat, 1989; Strauss et al., 2009; Billuart et al., 2008). It has a different thickness depending on regions, where average thickness at supero-inferior and antero-posterior directions are 9 mm and 5 mm, respectively (Halder et al., 2001; Howell and Galinat, 1989). Besides that, the mechanical

Corresponding author. E-mail addresses: [email protected] (A.H.A. Wahab), [email protected] (A.P.M. Saad), [email protected] (M.N. Harun), [email protected] (A. Syahrom), [email protected] (M.H. Ramlee), [email protected] (M.A. Sulong), [email protected] (M.R.A. Kadir). ⁎

https://doi.org/10.1016/j.jmbbm.2018.12.033 Received 14 August 2018; Received in revised form 18 December 2018; Accepted 20 December 2018 Available online 26 December 2018 1751-6161/ © 2018 Elsevier Ltd. All rights reserved.

Journal of the Mechanical Behavior of Biomedical Materials 91 (2019) 406–415

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properties of glenoid labrum also vary in various region. The highest compressive modulus of 0.4 MPa is found at superior-posterior region and the lowest compressive modulus can be seen at inferior region, 0.1 MPa (Carey et al., 2000). Since, there is no studies had been done to develop artificial glenoid labrum, however, the closest application to glenoid labrum that have almost similar properties is cartilage (Smith et al., 2008). Synthesizing of artificial cartilage used rubber-like materials such as Polyacrylamide (PAAm) (Dinu et al., 2007), Poly (2-hydroxy methacrylate) (PolyHEMA) (Fonseca Passos et al., 2011), polyvinyl pyrrolidone (PVP) (Katta, 2007), Poly(Acryclic acid) (PAAc) (Gulyuz and Okay, 2014), and polyvinyl alcohol (PVA) (Lozinsky, 2002; Hayes et al., 2016; Kim et al., 2015). Among them, PVA hydrogel shows a good characteristic in terms of biocompatibility, swelling properties, non-toxic and non-carcinogenic characteristics (Hassan and Peppas, 2000a). This shown by a sheer number of usage of PVA as an artificial cartilage (Curley et al., 2014; Kanca et al., 2018). Existing literatures has considered PVA as an artificial cartilage for the knee (Hayes et al., 2016; Curley et al., 2014; Hayes and Kennedy, 2016), hip (Yao et al., 1994), and shoulder (Święszkowski et al., 2006). The fascinating features of PVA is the ability to control its mechanical properties through manipulating concentration of polymer, time of freezing, time of thawing and number of freeze-thaw cycles (Hassan and Peppas, 2000a; De Rosa et al., 2014). Due to this advantage, PVA is a promising candidate for artificial glenoid labrum fabrication. The challenge in developing artificial glenoid labrum is due to different in compressive modulus of intact glenoid labrum at different region. Besides that, several techniques had been utilized in developing functionally graded hydrogel for biomedical applications, specifically for stem cell. The techniques includes diffusion (Lo et al., 2008), controlled dipping into crosslinking solution (Hopp et al., 2013), various photopolymerization process (Nemir et al., 2010), temperature gradients during crosslinking (Wang et al., 2012), and controlling the height of the substrate (Kuo Cheng-Hwa et al., 2012). However, several limitations of these conventional systems were figure out such as the toxicity from residual monomers, precursors, photoinitiators, and crosslinkers (Hao et al., 2011; Park et al., 2010); the complexity of fabrication processes and different surface chemistry from different densities of crosslinkers along the gradient (Hopp et al., 2013). Therefore, in this study utilising freeze-thaw method to synthesize the functionally graded hydrogel for artificial glenoid labrum. The advantages of this method as it could develop a hydrogel that could eliminate the toxicity due to chemicals, and without used specific instruments or difficult procedure (Kim et al., 2015; Oh et al., 2016). Conceptually, producing different stiffness of material especially using PVA hydrogel has been reported by Kim et al. (2015) for stem cell. They produce functionally graded PVA hydrogel using liquid nitrogen (LN2contacting gradual freezing and thawing method). Even this method could produce wide range of stiffness, however, it is difficult to produce specific stiffness in targeted region of hydrogel. Therefore, artificial glenoid labrum should be developed as functionally graded material in order to mimics properties of intact glenoid labrum. In addition, utilising the glenoid labrum features and function is crucial to minimize the glenoid loosening, especially due to RHP. From an extensive literature search, there are very limited reports regarding functionally graded hydrogel production for artificial cartilage. Nevertheless, the method producing functionally graded material has been done for stem cell, tendon-to-bone, osteochondral bone and etc (Kim et al., 2015). However, there is no functionally graded material used for artificial glenoid labrum with specific properties for shoulder replacement. Therefore, the aim of this study to synthesize a functionally graded artificial glenoid labrum from Polyvinyl Alcohol (PVA) hydrogel using a simple freeze-thaw method for shoulder replacement. The present work utilises a freeze-thaw method in synthesizing PVAbased hydrogel by controlling its concentration and number of cycles. Another advantage of using freeze-thaw method is a toxic chemicals involvement during the production can be avoided completely.

2. Materials and methods 2.1. Preparation of homogenous PVA hydrogel The PVA powder with molecular weight of 89,000–98,000 g mol-1 (> 99% hydrolyzed) was obtained from Sigma Aldrich (Sigma Aldrich, USA). Various amount of PVA powder, 10%wt, 15%wt, 20%wt and 25%wt were mixed with deionized water. In order to ensure the PVA powder dissolve completely, the solubilisation process was carried out by heating the mixtures in the oven for 30 min at 110 °C. Entrapped bubbles in the solution were removed by using vacuum pump (Rocker 300,Taiwan) before it can be poured into the mould. A complete freezethaw cycle was counted when the solution went through freezing and thawing processes. The solutions were stored in − 20 °C for 24 h for freezing process and continued with thawing process for another 24 h at a constant temperature of 30 °C. Freeze-thaw cycles were varied from one to six cycles for all four PVA concentrations (i.e.: 10%, 15%, 20% and 25%). 2.2. Preparation of functionally graded PVA hydrogel Prior to preparation of the functionally graded PVA hydrogel, the homogenous hydrogel were produced to determine the best configuration that match with compressive modulus of intact glenoid labrum. Then, the best configuration has been chosen by applying the following criteria: 1) the compressive modulus of PVA hydrogel must match with compressive modulus of intact glenoid labrum. Since this study was the first attempt to develop an artificial glenoid labrum with different stiffness gradient, therefore, only highest (0.4 MPa) and lowest (0.1 MPa) values had been considered (Carey et al., 2000). 2) If there was the same values of compressive modulus from two or more different PVA concentration in same FT cycles, thus, lowest PVA concentration would be prioritize than higher PVA concentration. 3) FT cycles that have both highest and lowest values of compressive modulus would be shortlisted and the lowest FT cycles would be selected. This criteria was important in order to ease the process of making the functionally graded PVA hydrogel and also to reduce the time consume for preparing and producing the hydrogel. A mould was used for preparing the functionally graded samples. Fig. 1 showed the actual mould and its schematic diagram. It was specifically designed and fabricated using 3D-printer (3D Systems Inc. USA). This mould basically had three main components, main body,

Fig. 1. Mould for functionally graded PVA hydrogel. 407

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caps, and divider. The divider used to divide the main body into tworectangular or three-cubic chamber. In preparing the functionally graded hydrogel, the PVA powder was mixed with deionized water and heat at 110 °C for 30 min to allow the PVA completely solubilized in deionized water and the bubbles trap in the solutions were removed using vacuum pump. The mould, initially, was divided into two rectangle chamber by placing the divider at the centre hole of top cap. Then, the solution with higher PVA concentration was poured into one side of the chamber, while, lower PVA concentration solution was poured at the other side. Pre-processing step was introduced into conventional freeze-thaw method, which includes pre-freezing and prethawing steps. In pre-freezing process, the mould with solutions was stored in the freezer at − 20 °C for two hours. Then, pre-thaw process was done by putting the solution in the oven with maintained temperature at 55 °C for 30 min. Then, the divider between two chambers was removed to allow diffusion of the solution from both sides. The solution left at room temperature for 15 min before went to its first freeze-thaw cycles.

Fig. 3. Stress-strain curve for PVA hydrogel. S1 obtained from between 1% and 15% strains.

where, ΔHm(i) was heat of melting of each section of PVA hydrogel determine from the DSC curve and ΔHm100% was heat of melting of 100% crystalline PVA, 138.6 J/g (Peppas and Merrill, 1976). Wide angle X-ray diffraction (WAXD) profiles was determine using XRD machine at room temperature, Rigaku SmartLab® X-ray Diffractometer (The Woodlands, TX). The operating tube voltage and current of the machine were 40 kV and 30 mA, respectively. The samples were scanned at the rate of 0.04° 2θ s−1 in the 2θ range 10–40°. This 2θ range had been choose by referring to previous study had been conducted for PVA hydrogel that indicate the crystalline peak of PVA occurs at 19.4° 2θ (Ricciardi et al., 2004; Holloway et al., 2013a). Three samples representing each region of high concentration, integration area and low concentration area were analysed using WAXD. The crystallinity for all three samples were calculated using Eq. (2).

2.3. Uniaxial compression test Uniaxial compression test was conducted for all configuration of PVA hydrogels (Fig. 2) using universal testing machine (The FastTrack 8874, Instron, Norwood, USA) with capacity of load cell was 25 kN. Unconfined compression test were conducted on the samples of hydrogels with dimension 21 mm × 21 mm × 21 mm. Cross head speed used was 1.33 mm/min exerted until 40% of strain. The elastic modulus of the hydrogel was obtained from first linear slope, range between 1% and 15% strains, of stress-strain diagram as shown in Fig. 3. In addition, the modulus was calculated for 24 configurations, where four different concentrations (10%, 15%, 20% and 25%) would have six different number of freeze-thaw cycles (one to six freeze-thaw cycles). All 24 configurations was repeated at least three time (n = 3) for homogenous hydrogel. Whilst, for functionally graded samples, at least 10 independent samples (n = 10) were tested.

% of crystallinity=

(2) Crystalline peak area had been determine from the PVA crystalline peak 19.4° area and it was divided with total area that covered both crystalline and amorphous peak area, begin from 10° to 40°. The microstructure of the PVA hydrogel had been observed using scanning electron microscopy (SEM) (TM3000, Hitachi) at an accelerating voltage 15 kV. Swelling behaviour of each sample was obtained by immersing the sample in Phosphate Buffer Saline (PBS) solution (pH 7.4) at constant temperature 37 °C. A weight of the swollen hydrogel was determined every two days. The hydrogel was blotted on filter paper to remove adsorbed water at the surface then it was weighed immediately on electronic balance. The swelling ratio (Q) was calculated using Eq. (3)

2.4. Materials characterization Melting temperature, Tm, and heat of melting, ΔHm, of the functionally graded PVA hydrogel had been determined using a differential scanning calorimeter (DSC) (Perkin-Elmer DSC 7, USA) for each sections of high concentration area, intermediate area, and low concentration area. The hydrogel was cut from each section, which weight between 10 and 15 mg, and sealed in an aluminium pan. Then the specimen was heated from 25 °C to 250 °C at 10 °C/min rate. Based from DSC curve, the Tm and ΔHm were directly determined. The percentage of the crystallinity of PVA for each sections were calculated using Eq. (1):

% of crystallinity=

Q=

Ws

Wd Wd

× 100

(3)

where, Ws was weight of hydrogel during swollen state and Wd was weight of hydrogel in dry state. Three independent samples were tested for each set of the hydrogels (n = 3).

Hm(i) Hm100%

Crystalline peak area × 100 Crystalline peak area + Amorphous peak area

(1)

2.5. Finite element analysis (FEA) 3D model of scapula bone was reconstructed from CT data images using Mimics software (Materialise, Leuven, Belgium). The slide thickness of CT data was 0.537 mm. Next, glenoid implant, cement and glenoid labrum were constructed using computer aided design (CAD) software (SolidWorks; Dassault Systèmes SolidWorks Corp., Concord, MA, USA). Implant thickness, type of fixation, and radius of curvature of glenoid implant were set to 4 mm, peg-fixation, and 29.5 mm, respectively. Cement thickness was set as 0.5 mm (Terrier et al., 2005). Two type of implant were constructed, conventional all-polyethylene implant and gleno-labrum implant. Virtual surgery were done by combining all parts together in Mimics software. Then, remeshing for

Fig. 2. Compression test of PVA hydrogel. 408

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all parts were done using element size determined from convergence study. Total elements and nodes for glenolabrum case were 405,000 and 161,000, respectively. In conventional cases, the total element and nodes were 338,000 and 87,000. Cortical bone was assigned as isotropic material with modulus 16 GPa and Poisson's Ratio 0.3. Whille, cancellous was assigned as orthotropic material where E11 = 342.1 MPa, E22 = 212.8 MPa, E33 = 194.4 MPa, v12 = v13 = v23 = 0.26, and G12 =G13 = G23 = 100 MPa. Modulus for implant was 0.965 GPa with Poisson's ratio 0.34 and modulus for cement was 2.2 GPa and Poisson's ratio 0.23 (Mansat et al., 2007). Hyperelastic equation for artificial glenoid labrum was determined from experimental data using experimental data fit tools available in MSC Marc Mentat software (MSC Software, Santa Ana, USA). The third order deformation hyperelastic model was found to describe the best for PVA hydrogel based on the good agreement between calculated stress-strain relation and the experimental data. The equation for this model was described as in Eq. (4):

C01 (I2 3) + C11 (I1 3)(I2 3) + C20 (I1 3)2 1 + C30 (I1 3)3 + (J 1)2 Di

cartilage (Curley et al., 2014). In previous studies, it was considered as artificial cartilage for the knee (Hayes et al., 2016; Curley et al., 2014; Hayes and Kennedy, 2016), hip (Yao et al., 1994) and shoulder cartilage (Święszkowski et al., 2006). PVA hydrogel could be formed by creating physical crosslinks using freezing and thawing process. Mechanical properties of PVA could be tailored by changing PVA concentrations, number of FT cycles, freezing time, and thawing time (Holloway et al., 2013a; Lozinsky et al., 2003). PVA gelation begin when the PVA-water solution was frozen under temperature below crystallization point. During this process, the solution was separated into two-phase, PVA-rich region and unfrozen liquid microphase (UFLMP), which in this region it has poor PVA and high water content. In PVA-rich region, PVA chains were closer to each other which encouraged the formation of crystallite and hydrogen bond (Holloway et al., 2013a). Moreover, water in UFLMP formed as an ice blocks and it became as a base for formation of macropores which will describe later. Once completing freezing process, the process continued with PVA mixture was thawed at room temperature. During this process, the ice blocks from UFLMP melted and formed as macropores which filled with solvent. While, the formation of gel from crystallite and hydrogen bond during freezing process did not melted and remains as gel during thawing process (Lozinsky, 2002; Lozinsky et al., 2003). Repeating this freezing and thawing process would increase the formation crystallite and hydrogen bonding between PVA chains. Our results was in good agreement with the theory where increasing the concentration of PVA and FT cycles caused increased of PVA compressive modulus (Lozinsky et al., 2003). Concept of gel-formation was illustrated in Fig. 5. In the diagram, (1) refers to PVA monomers in the solution; (2) water in the solution; (3) PVA-rich region; (4) ice blocks which act as pore-agent; (5) gel formed during freezing process; (6) macropores consisting water formed after ice block melt during thawing process. Glenoid labrum was a passive stabilizer and contribute about 10% of stability (Halder et al., 2001) for glenohumeral joint by deepen the glenoid curvature up to 50% (Howell and Galinat, 1989). In addition, this passive stabilizer absorb the stress induce from humeral head translation and limit the translation of humeral head to ensure the humeral head remain in the centre of glenoid curvature (Howell and Galinat, 1989; Strauss et al., 2009; Billuart et al., 2008). During total shoulder replacement, this component had been excised and, unfortunately, conventional implant had been used to replace glenoid ignored the function of glenoid labrum. As a consequences, the high stress at edge of glenoid implant during eccentric loading had been frequently reported (Zhang et al., 2013; Geraldes et al., 2016) and it had been associated with glenoid component loosening due to RHP (Matsen et al., 2008; Armstrong and Lewis, 2013). Compressive moduli for intact glenoid labrum varies between 0.1 MPa and 0.4 MPa (Carey et al., 2000). Thus, three criteria had been underline in order to determine the best configuration of PVA hydrogel to be used in developing artificial glenoid labrum. Firstly, the compressive modulus of PVA hydrogel must match with compressive modulus of intact glenoid labrum. Since this study was the first attempt to develop an artificial glenoid labrum with different stiffness gradient, therefore, only highest (0.4 MPa) and lowest (0.1 MPa) values had been considered. Second, if there was the same values of compressive modulus from two or more different PVA concentration in same FT cycles, thus, lowest PVA concentration would be prioritize than higher PVA concentration. Third, FT cycles that have both highest and lowest values of compressive modulus would be shortlisted and the lowest FT cycles would be selected. These criteria were important in order to ease the process of making the functionally graded PVA hydrogel and also to reduce the time consume for preparing and producing the hydrogel. Table 1 showed the configuration that fit three criteria as mention above for all 24 configurations available. In the table, configuration that fit with first, second and third criteria had been label as C1, C2, and C3 respectively. In addition for first criteria, configuration that meet

W = C10 (I1 3) +

(4)

where W is the strain energy density; Cij is material constants that control shear behaviour; Di is material constant that control bulk compressibility; J elastic volume ratio and I1,I2 are the first and second invariants of the deviatoric strain respectively (Święszkowski et al., 2006). Since we assume the hydrogel as incompressible, therefore, the materials parameter Di is equal to zero. The other material parameters was determined automatically from the software for this model are: C10 = 1.29; C01 = -1.31; C11 = -5.61; C20 = 4.19; C30 = -0.01. The model was fixed at medial scapula and 750 N was applied at implant in three different location, which are centre (C), superioranterior (SA) and superior-posterior (SP). This load representing the daily activities of elder people such as standing and sitting from chair, walking with stick and lifting a 5 kg bag (Anglin et al., 2000). Contact at implant-bone and cement-bone was assigned as non-bonded with friction coefficient 0.6 (Wahab et al., 2017) to allow micromotion at the interface. The finite element analysis was conducted using MSC Marc Mentat software. Several mechanical parameters such as stress at the implant, micromotion at cement-bone interface and implant-bone interface were computed. 3. Results and discussion 3.1. Homogenous PVA hydrogel First step in developing functionally graded PVA hydrogel for artificial glenoid labrum was developing homogenous PVA by varying the concentration of PVA and number of FT cycles. It was crucial to ensure the properties of PVA hydrogel suit to intact glenoid labrum properties. Therefore, the mechanical properties for homogenous PVA hydrogel was done in order to determine the best configuration that fit to intact glenoid labrum. Finding from this study showed that, the mechanical properties for homogenous PVA hydrogel had increasing trend as PVA concentration and the number of FT cycles increased. In low concentration (10% PVA), the small inclination of stiffness has been recorded and it remained the same at third FT cycles and above. On the other hand, for high concentration (25% PVA) a significant increase in hydrogel stiffness had been recorded and it still increasing even for sixth FT cycles. Fig. 4 showed the mechanical properties of homogenous PVA hydrogel for four different concentration and six FT cycles. PVA hydrogel become favourable as biomaterial due to its swelling properties, biocompatibility, non-carcinogenic and non-toxic (Hassan and Peppas, 2000a). Moreover, ability of PVA in manipulating its mechanical properties make it as a popular choice for tissue engineering (Jiang et al., 2011). For instance, it had been widely used as an artificial 409

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Fig. 4. Mechanical properties of homogenous PVA hydrogel for 24 configurations.

with lower compressive modulus, 0.1 MPa, of glenoid labrum had been annotated with subscript L, while, subscript H represent the configuration which fit with higher compressive modulus, 0.4 MPa, of glenoid labrum (Carey et al., 2000). For first criteria, configurations that fit to lowest compressive modulus of glenoid labrum were FT1–20%, FT2–15%, FT3–10%, FT4–10% and FT5–10%. While, FT3–20%, FT4–15%, FT5–15%, FT5–20%, and FT6–15% suits for highest compressive modulus of glenoid labrum. Since, in FT5, there were two configurations that fit to higher compressive modulus, thus, based on second criteria, configuration with 15% PVA had been selected and considered for the last criteria. There were three FT cycles that fulfil third criteria, which are FT3, FT4, and FT5. One complete FT cycles consume two days for preparing the samples, therefore, FT3 was favourite as optimize configuration as it has consumed lowest time for preparing the samples as compared to other FT cycles that fulfil other criteria.

Table 1 Configurations of PVA hydrogel for artificial glenoid labrum. FT cycles/Concentrations FT FT FT FT FT FT

1 2 3 4 5 6

10%

C1L;C3 C1L;C3 C1L;C3

15% C1L C1H;C3 C1H;C2;C3 C1H

20%

25%

C1L C1H;C3 C1H

Note: C1L: Criteria 1 (Lowest Compressive Modulus); C1H: Criteria 1 (Highest Compressive Modulus); C2: Criteria 2; C3: Criteria 3.

3.2. Functionally graded PVA hydrogel In this mechanical testing, 10 samples underwent for compressive testing, which each sample had been divided into three segment and labelled with high-PVA, intermediate-PVA, and low-PVA regions. Compressive modulus for functionally graded PVA increase gradually from lower to higher concentration region. In higher-PVA region, mean compressive modulus was 0.41 ± 0.054 MPa, while, in lower-PVA concentration region, mean modulus was 0.10 ± 0.031 MPa. Mean modulus for intermediate-PVA region was in between higher and lower region, 0.21 ± 0.059 MPa. Fig. 6 show compressive modulus of PVA hydrogel obtained from stress-strain diagram. In order to confirm the samples had similar modulus with intact glenoid labrum, results from compression test showed the high-PVA region had 0.4 MPa and lowPVA region had 0.1 MPa for compressive modulus which fit to highest and lowest modulus of intact glenoid labrum (Halder et al., 2001). Moreover, the intermediate-PVA region records a compressive modulus 0.21 MPa which lies in between high-PVA and low-PVA regions,

Fig. 6. Functionally graded polyvinyl alcohol hydrogel.

simultaneously prove the existence of gradual stiffness at the sample. Since, the number of FT cycles, time for freezing and thawing remain constant, therefore, the formation of various stiffness was depends on the amount of PVA chains exist at each region. Higher stiffness was observed when PVA concentration increase due to intense PVA chains in the solution which create more PVA-rich region (Holloway et al., 2013b). High intensity of PVA-rich regions promotes greater formation of physical crosslinks in term of crystallites and hydrogen bonding (Holloway et al., 2013a). Fig. 7 showed the melting point of PVA hydrogel, ~ 230 °C, for all

Fig. 5. Conceptual diagram of hydrogel formation process. 410

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Fig. 8. WAXD curve. Fig. 7. DSC curve for functionally graded PVA hydrogel.

this region as compared to intermediate-PVA and low-PVA region. The peak of crystalline for PVA hydrogel in this study was found at 19.23° 2θ, it correspond to a d spacing of 4.61 Å. Additionally, and it was in line with previous studies that mention the peak located at about 19.4° 2θ (Ricciardi et al., 2004; Watase and Nishinari, 1985). This peak was acknowledge as 101 reflection from PVA crystal in monoclinic form, which indicate the PVA hydrogels are crystalline (De Rosa et al., 2014; Willcox et al., 1999). Moreover, the gradually decreasing percentage of crystallinity became as strong evidence, since it was consistent with another two previous tests (compression test and DSC), that show the gradual formation of crystallites along the PVA hydrogel sample. Turning now to the microstructure of functionally graded PVA hydrogel, Fig. 10 showed microstructure for three different region, highPVA, intermediate-PVA and low-PVA regions. Pore in low-PVA region could be seen clearly and the visibility of pore were gradually decrease towards high-PVA region. Microstructure of PVA hydrogel was changed when concentration of PVA and FT cycles had been manipulated (Holloway et al., 2013a). In Fig. 10, SEM image showed decreasing the concentration of PVA result in formation of bigger pores. As mentioned earlier, the formation of physical crosslinks have direct relationship with intensity of PVA. Higher intensity of PVA promotes more crystallite formation which also form denser polymer mesh. As a consequence, pore size of the sample becomes smaller because most of the regions were PVA rich regions which created thicker PVA-rich wall (Millon et al., 2007). Fig. 11 illustrate the formation of pore due to different concentration of PVA. In addition, the concept of gel formation, as stated in Fig. 5 previously, also could support the explanation for formation of different sizes of pores due to increase in PVA concentration. Based on the concept, increased PVA concentration caused decreased water content in the initial PVA solution. Thus, reduction in water content lead to formation smaller ice blocks during freezing process, where this ice blocks act as pore-agent (Dinu et al., 2007; Lozinsky et al., 2003). Therefore, the microstructure obtained support previously crystallinity results. Moreover, denser polymer mesh and higher amount of crystallite created in high-PVA regions result stronger mechanical strength of PVA hydrogel in term of its compressive modulus. Swelling characteristics of PVA hydrogel was another important aspect to develop suitable material for soft tissues applications, in this study, glenoid labrum. The hydrogel was immersed in PBS solution for conducting swelling analysis (Shi and Xiong, 2013). Swelling behaviour of PVA seems to have significant relation with concentration of PVA, where, increase in concentration of PVA could reduce the ability of hydrogel to store water. Table 3 show that with Low-PVA region had ability to store the solution two-fold higher compared to High-PVA region. Whilst, the swelling behaviour at the intermediate-PVA region lies in between high-PVA and low-PVA indicate that, the gradual decrease of swelling ratio on the material. This is important because the abrupt change on material properties could be the weakest point of the material itself which lead to material failure. This results agree with the theory, where region with higher PVA content produced smaller pore size. Thus, only small portion of water could store in the hydrogel structure. On the other way, low PVA content region had bigger pore

three regions of functionally graded PVA hydrogel, high-PVA, intermediate-PVA and low-PVA. High-PVA region had highest Tm with 230.57 °C, while, intermediate-PVA and low-PVA region had 229.97 °C and 229.90 °C respectively. This increasing trend also had been observed for heat of melting, ΔHm, from low-PVA (2.1980 J/g) to highPVA (4.1377 J/g) region. Information of ΔHm was used to calculate percentage of crystallinity for functionally graded PVA at each region using Eq. (1), as stated previously. The percentage of crystallinity for all three regions were stated in Table 2. The crystalline peak for functionally graded PVA hydrogel was located at 19.23° ± 0.04° 2θ, Fig. 8. Crystallinity at each region was determined by dividing the area under PVA crystalline peak with total area of PVA crystalline peak and amorphous peak, range within 2θ 15–35°, as in Table 2. Highest crystallinity found in high-PVA region (6.43%), followed by intermediate-PVA (6.17%) and lowest crystallinity was observed in low-PVA region (4.59%). In addition, two-dimensional WAXD patterns, Fig. 9, showed the red line (in black ring) was thickest in high-PVA region followed by intermediate-PVA region. While, the red line was almost faded in low-PVA region. Besides mechanical test, the existence of gradual stiffness in the hydrogel was supported by results of DSC and WAXD, where the crystallites formation was differed at each region. DSC curves provide thermal properties information such as melting temperature (Tm) and heat of melting (ΔHm). The latter properties values had been used to calculate the crystallinity of functionally graded PVA hydrogel at each region using Eq. (1) (Peppas and Merrill, 1976; Hassan and Peppas, 2000b). Decreasing trend from high-PVA region towards low-PVA region for Tm and ΔHm obtained from DSC curves and percentage of crystallinity was consistent with our mechanical experiment results which reflect the intensities of crystallites formed at each region. A possible explanation for this trend was, due to high crystallite formation, higher temperature needed to break the bond between PVA chains and more heat required for melting, ΔHm, the PVA (Watase and Nishinari, 1985). In contrast, at the low PVA region, lower temperature and smaller heat were required to melt the sample due to weak bonding which simultaneously indicate less crystallite formation at that region. Further investigation on crystallinity of functionally graded PVA hydrogel was done using WAXD. Diffraction from crystallite in the hydrogel formed a diffraction ring in 2D WAXD as showed in Fig. 9. Thickest diffraction ring (red line in the black ring) was found in highPVA region indicating the existence of greater amount of crystallite in Table 2 Percentage of crystallinity of functionally graded PVA hydrogel. Region

High-PVA Intermediate-PVA Low-PVA

Percentage of crystallinity DSC

WAXD

2.99% 1.93% 1.58%

6.43% 6.17% 4.59%

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Fig. 9. Two dimensional WAXD pattern for three different region.

Fig. 11. Concentration of PVA changed pore size of hydrogel. Table 3 Swelling behaviour of PVA hydrogel at three different region. Region

High-PVA

Intermediate-PVA Low-PVA

Swelling Ratio, Q (%)

364% ± 16.6% 414% ± 47.8%

801% ± 22.3%

absorb the stress exerted at this side. Additionally, for soft tissue application, limited volume change was desirable, to ensure this material could fit the size of tissue had been replace and avoid significant change on it mechanical properties (Holloway et al., 2011). 3.3. Simulation results Current conventional implant which made from Ultra-HighMolecular-Weight Polyethylene (UHWMPE) had two disadvantages, first, the design was like a “plate” which would increase humeral translation and produce eccentric load (Billuart et al., 2008; Sins et al., 2012). Second, it was insufficient to mimic the viscoelastic properties of cartilage and glenoid labrum which function as absorber for glenohumeral joint from high compressive load from humeral head (Halder et al., 2001; Strauss et al., 2009; Święszkowski et al., 2006). This advantages contribute to the most common complication after total shoulder arthroplasty which is glenoid component loosening. Therefore, it was crucial to restore back the function of glenoid labrum in current implant, thus, component loosening problem could be reduce. Fig. 12 showed results for stress exerted at the implant, during C load, both implant, standard and glenolabrum implant, were stable as the stress exerted at the implant was lower than 8 MPa. However, when the implant was loaded with eccentric load, the stress exerted at the

Fig. 10. Microstructure of PVA hydrogel at each region.

size which increase the ability of hydrogel to swell by allowing the solution to store in the hydrogel (Gupta et al., 2012). In addition, this increasing swelling ratio from high-PVA to low-PVA region was a sign of the existence of different pore size which related to the ability of the material to swell was gradually increase from high-PVA to low-PVA region. Furthermore, a number of previous studies reported that, for movement of shoulder joint (specifically glenohumeral joint) for most of daily activities, the humeral head was located at the superior region (Massimini et al., 2014; Shapiro et al., 2007). Therefore, highest stiffness should be located at the superior region to allow the material 412

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Fig. 12. Stress distribution at glenoid implant.

Fig. 13. Micromotion at cement-bone interface.

implant increase for both implant. As compared to C load, standard implant records almost five times higher (24 MPa), while, the stress was double for glenolabrum implant (12 MPa). In many previous study (Rockwood et al., 2009; Matsen et al., 2008; Anglin et al., 2001), high stress at the rim of implant during eccentric loading could lead to glenoid implant loosening due to RHP. This high stress not only caused implant wear, however, induced micromotion at the interfaces (Terrier et al., 2005, 2012) as well as high stress at the cement mantle (Terrier et al., 2012). Our findings showed that standard all-polyethylene implant was exposed to implant failure especially during eccentric load and it in agreement with other previous study that eccentric load produced high stress a the implant (Wahab et al., 2017, 2016; Hadi et al., 2015). Interestingly, new glenolabrum implant was superior compared to current standard all-polyethylene implant since it had lower implant stress, up to 51%, when loaded with eccentric load. The artificial glenoid labrum from PVA hydrogel could act as an absorber (Święszkowski et al., 2006) to reduce the stress level at the implant by absorbing the stress when the glenoid implant had been loaded with eccentric load. Besides that, existence of artificial glenoid labrum could prevent from implant wear, which induced by high stress at the implant. Micromotion at interface become another indicator of glenoid component loosening due to RHP. In various previous studies (Anglin et al., 2001; Wahab et al., 2016; Pomwenger et al., 2015), micromotion at the interface had been assessed to measure the performance of glenoid implant. Two interfaces had been assessed which are cement-bone interface and implant-bone interface. Fig. 13 showed glenolabrum implant had 13% and 17% lower micromotion at cement-bone interface compared to the standard implant during SA and SP loadings, respectively. Micromotion at implant-bone interface also showed glenolabrum implant having lower micromotion compared to the standard implant, as per Fig. 14. In SA and SP loads, the glenolabrum implant was 17%

Fig. 14. Micromotion at implant bone interface.

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and 15% less than the standard implant, respectively. Despite the fact that during concentric load, the glenolabrum implant had higher micromotion at the inferior side, 16 µm, than the standard implant, it was lower than the threshold value of 20 µm. Several limitation had been noted in this study which could be improve in future studies and produce better outcome. First, this study divided the functionally graded PVA hydrogel into three regions only. However, in this study, we believe it was enough to prove the existence of gradual stiffness in functionally graded PVA hydrogel to be used as artificial glenoid labrum. Secondly, the functionally graded PVA hydrogel was prepared with 20% and 10% PVA concentration under three FT cycles only. Therefore, it was quite difficult to differentiate the pore sizes between this three regions from SEM images. The formation of pores had been associated with PVA concentration and FT cycles (De Rosa et al., 2014; Holloway et al., 2011). Relation between pore sizes and PVA concentration already had been discussed. Increased FT cycles would change the microstructure of PVA hydrogel. In each cycles, freezing water expelled PVA which produced a regions which dense with PVA. This mechanism made the PVA chains closer to each other and encouraged the formation of crystallites and hydrogen bond, simultaneously, formed a thinner wall of PVA-rich region after certain FT cycles (Holloway et al., 2013a). Thirdly, biodegradability of hydrogel was not conducted in the present study, since aim of this study to determine the mechanical properties of PVA that suit to glenoid labrum. Current state, the mechanical properties was suit to artificial glenoid labrum, which could reduce the stress exerted at the glenoid implant. However, for enhancement of artificial glenoid labrum, future studies could focus on maintaining its properties and improve the longevity of the material. Alves et. al had been reported that PVA hydrogel with five functional group could degrade about 50% in 4 months (Alves et al., 2012). Theoretically, hydrogel that have loose crosslink form a fragile network, thus, this network could be easily to break and it accelerate degradation of the material (Okay and Lozinsky, 2014; Martens et al., 2002). While, in term of mechanical properties, this fragile network have low mechanical properties. In contrast, hydrogel that had intense crosslink will form stronger network. Thus, extra force needed in order to break the bonding between networks. As consequence, this would decelerate the degradation of material and prolong the life of the material. Several factors influence the degradation rate of the material, includes main chain polymer, crosslink densities (amount, type and size of crosslinking molecules) (Drury and Mooney, 2003), degree of hydrolysis (Solaro et al., 2000) and environmental conditions (Drury and Mooney, 2003). Numerous studies had been conducted to improve the material properties of hydrogel using various technique such as doublenetwork (Gu et al., 2018), gel made by mobile crosslinkers (Haraguchi and Takehisa, 2002), topological gels (Noda et al., 2014), gels form by hydrophobic associations (Tuncaboylu et al., 2011) and microsphere composite hydrogels. Last but not least, in this study, only static load with three different location had been applied to the glenoid implant. This may underestimate the potential of functionally graded PVA hydrogel as artificial glenoid labrum. However, based from this static analysis, the existence of this component in conventional glenoid implant was promising. It was due to ability of the implant to reduce stress at implant and micromotion at the interfaces during eccentric load.

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